L-arabinose

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 06, 2017 - July 20, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP

Version / remarks:

10 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Toxi-Coop ZRT. 8230 Balatonfüred, Arácsi út 97. (1045 Budapest, Berlini út. 47-49.) Hungary

Test material

Test material information

Constituent 1

Reference substance name:

L-arabinose

EC Number:

226-214-6

EC Name:

L-arabinose

Cas Number:

5328-37-0

Molecular formula:

C5H10O5

IUPAC Name:

L-arabinose

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: AD16081001
- Expiration date of the batch: 2019-08-09

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Stable under ambient conditions

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The EpiOcular™ human cell construct (MatTek Corporation) is used in this assay. This three-dimensional human cornea model allows identification test items potential to induce eye irritation or serious eye damage by assessing cell viability after treatment. The model is composed of stratified human keratinocytes in a three-dimensional structure, consisting of at least three viable layers of cells. Test materials can be applied topically to the model so that also water insoluble materials may be tested. Prior to use, each plate (6, 12, and 24-well) and its cover will be uniquely identified with a permanent marker by a plate number and/or test article number.
The cytotoxicity of the test article (and thus the ocular irritation potential) is evaluated by the relative viability of the treated tissues in comparison to the negative control-treated tissues. Viability is determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control and test article-treated cultures (Berridge, et al., 1996). Data are presented in the form of relative survival (relative MTT conversion). The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 μL of 0.3 % (v/v) Triton X-100).
For further information see "Any other information on materials and methods.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

25 min post-soak phase
18 h post-treatmnt incubation
3 h MTT-incubation

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used:
After the test kit arrival, the tissues were equilibrated to room temperature for about 15 minutes. The Assay Medium was pre-warmed to 37 °C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37 °C in an incubator with 5 % CO2, 90 ± 10 % humidified atmosphere for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours). After the overnight incubation, the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37 ± 1 °C in an incubator with 5 ± 1 % CO2, 90 ± 10 % humidified atmosphere).

- RhCE tissue construct used, including batch number:
EpiOcular™ (OCL-200-EIT), Lot No: 23785, MatTek In Vitro Life Science Laboratories Mlynské Nivy 73, 821 05 Bratislava, Slovakia

- Doses of test chemical and control substances used:
Test substance: 50 mg
pos./neg. Control: 50 µL

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
25 min post-soak phase
18 h post-treatmnt incubation
3 h MTT-incubation

- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: 570 nm

- Description of the method used to quantify MTT formazan:
Following the formazan extraction, 200 μL sample(s) from each tube (2×200 μL) was placed into the wells of a 96-well plate (labelled appropriately) and read Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm using isopropanol solutions as the blank (8×200 μL).

- Complete supporting information for the specific RhCE tissue construct used: See "Any other information on materials and methods"

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals:
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392-492-1722) using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.

- Positive and negative control means and acceptance ranges based on historical data:
See "Any other information on materials and methods"

Results and discussion

In vitro

Results

Irritation parameter:

other: % cell viability

Value:

87

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Validity of the test:

The mean OD value of the two negative control tissues was: 1.997 The positive control result showed 7 % viability at 6 hours exposure. The highest difference of viability between the two tissue replicates: 14 %. All validity criteria were within acceptable limits in the experiment therefore the study can be considered as valid.

Indicator for potential false viability:

Possible direct MTT reduction with test substance: No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test substance: The test item showed no ability to become coloured in contact with water and isopropanol. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells are presented below:

Test Material Optical Density (OD) Viability (%) Δ% Neg. Control 1 1.862 93 14   2 2.133 107   Mean 1.997 100   Pos. Control 1 0.070 4 8   2 0.222 11   Mean 0.146 7   Test Substance 1 1.703 85 4   2 1.785 89   Mean 1.744 87     Standard deviation (SD) 2.89

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro eye irritation assay according to OECD Guideline 492, the test substance did not shown eye irritating properties.

Executive summary:

In an in vitro eye irritation assay according to OECD Guideline 492, the acute ocular irritation potential of the test item L-Arabinose on three-dimensional RhCE tissue in the EpiOcular™ model was determined.

Before treatment the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 °C in an incubator with 5 % CO2, 90 ± 10 % humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test items treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 ± 1 °C in an incubator with 5 ± 1 % CO2 protected from light, 90 ± 10 % humidified atmosphere. The formazan precipitated was then extracted using isopropanol and quantified spectrophotometrically. Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated 6 hours ± 15 minutes. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60 % of the negative control. Depending on the regulatory framework in member countries, the test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %. The test item L-Arabinose did not show significantly reduced cell viability in comparison to the negative control (mean viability: 87 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to eye.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.